The object of the proposed research is to analyze the mechanism by which the (Ca plus Mg) ATPase (E) of cardiac muscle transports calcium into the interior of the sarcoplasmic reticulum (SR) and to discover why that mechanism is depressed in the failed myocardium. METHODS: 1) Vesiculated sarcoplasmic reticulum (SRV) will be prepared by differential centrifugation. 2) E will be purified from the SRV with detergents and sucrose gradients. 3) A nondenatured stable phosphoprotein (EP) will be prepared by incubating E with ATP plus Ca2 ion followed by exposure to EDTA. 4) The ionophoric activity of EP will be measured in a double sector cell. 5) Latent E will be defined and measured as that E within the SRV which is not phosphorylated by ATP unless detergent is present. 6) The rate of EP formation will be studied by a chemi-quench technique. 7) Cardiac failure will be produced in canine heart-lung preparation. OBJECTIVES: 1) Isolate and purify the SR (Ca plus Mg) ATPase. 2) Determine the most probable form of E involved in calcium transport by measuring calcium binding to E, EMg, EMgNTP (the magnesium and nucleoside triphosphate form of the enzyme) and EP. 3) Determine the availability of the calcium binding site of E when E is in the membrane and is in one of the following forms: E, EMg, EMgNTP or EP, by subjecting SRV to tryptic digestion. With E in each of these forms embedded in the membrane matrix, the formation of ionophore complexes will be measured. 4) Determine the step(s) in ATP hydrolysis and calcium transport which are depressed in SRV from failed myocardium. Three major areas of the ATP hydrolysis mechanism will be studied: a) the ratio of latent to active enzyme concentration, b) the rate of EP formation, and c) the rate of EP breakdown.